DNAs form a double-stranded structure though base pairs A-T and G-C. The formation of a double strand by hybridization of DNAs due to complementarity of base pairs is applied to various fields such as a DNA tag for detecting DNA, a DNA probe for, for example, Southern hybridization, a DNA array, a zip-code of DNA, real-time PCR technologies such as TaqMan, a molecular beacon, a DNA affinity column for isolating DNA having a specific sequence, and also a DNA computer. The number of sequences composed of four types of bases, A, G, C, and T, is enormous, e.g., about 106 (=410) even in the case of a DNA consisting of only 10 bases. However, even if a wrong base pair other than A-T or G-C (mismatched base pair such as G-T) is present at one position of the 10-base DNA, a double strand may be formed (mishybridization) under some conditions, which may lead to incorrect DNA detection. In particular, since a G-C base pair is thermally stable than an A-T base pair, the stability of double-stranded DNAs varies depending on a difference in content of the G-C base pairs even if the DNAs have the same length. A double-stranded DNA having a high G-C content, even if the DNA has one mismatched base pair, the stability thereof tends to be higher than that of a double-stranded DNA having a low G-C content. Hybridization in practical technologies is performed under the same temperatures and the same conditions. Accordingly, in order to use a plurality of DNA tags or probes, it is important that the respective double-stranded DNAs have the same thermal stability. Mishybridization in such a method using a plurality of DNA tags or probes can be reduced if the variation of base pairs is increased by using a novel unnatural base pair in addition to base pairs, A-T and G-C, and a constant thermal stability is achieved regardless of the G-C contents of different double-stranded DNAs having the same length by using the novel unnatural base pair. The accuracy in each practical technology can be thereby enhanced. Furthermore, the accuracy of a method such as SNP analysis can be enhanced if a probe that can selectively distinguish between match and mismatch with a target DNA at a single-base mutation level is produced.
The technology for expanding genetic information of DNA by unnaturally forming novel base pairs has two possible applications having high versatility, and unnatural base pairs have been actively being developed. One of the applications is formation of an unnatural base pair that functions in replication, transcription, or translation to produce DNA, RNA, or protein containing a novel constituent. The other application is formation of a double-stranded nucleic acid by incorporating an unnatural base pair into DNA or RNA to increase the number of nucleic acid fragment probe sequences, which can be applied to a multiplex by real-time PCR and a DNA computer and further can be used in a novel codon and anticodon in introduction of an unnatural amino acid into protein by translation.
S. A. Benner, et al. produced base pairs such as isoG-isoC by modifying the combination of hydrogen bond patterns in natural base pairs and found that these unnatural base pairs selectively form base pairs in a double-stranded DNA (Patent Documents 1 to 3 and Non-Patent Documents 1 and 2). J. R. Prudent, et al. have produced a novel DNA probe using an isoG-isoC base pair and have designed a multiplex PCR method (Patent Document 4 and Non-Patent Documents 3 to 6). However, these base pairs have hydrogen bonding substituents; hence, amidite reagents in which protecting groups are introduced to the substituents of the bases are necessary to be prepared for DNA synthesis. The present inventors have intensively developed the third, unnatural base pairs (unnatural base pairs) for expanding genetic information of DNA and have successfully developed several unnatural base pairs that function in replication or transcription, such as an s-y base pair (s: 2-amino-6-thienylpurine, y: pyridin-2-one), a v-y base pair (v: 2-amino-6-thiazolyl purine), an s-Pa base pair (Pa: pyrrole-2-carbaldehyde), a Ds-Pa base pair (Ds: 7-(2-thienyl)-imidazo[4,5-b]pyridine), and a Ds-Pn base pair (Pn: 2-nitropyrrole) (Patent Document 5 and Non-Patent Documents 9 and 10). However, since these unnatural bases are utilized as base pairs, amidite reagents of two types of bases are necessary to be prepared.
In order to overcome these disadvantages, if a base not having hydrogen binding substituents self-complementarily forms a base pair, a preparation of only one amidite reagent may be sufficient, which significantly facilitates the use of the bases. F. E. Romesberg, et al. have developed such a self-complementary base pair (Non-Patent Documents 7 and 8). Their self-complementary base pair (7-propynyl isocarbostyril) is more stable than the G-C base pair, and the stability of a base pair with a natural base is reduced by 7 to 11° C. Though this unnatural base is self-complementarily incorporated into DNA by replication, the replication stopped after formation of the self-complementary base pair.
Further, in unnatural base pairs that have been already developed, thermal stability of DNA in each combination of an unnatural base pair and a natural base pair adjacent to each other has not been comprehensively investigated.